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Applied and Environmental Microbiology, December 2007, p. 7753-7756, Vol. 73, No. 23
0099-2240/07/$08.00+0     doi:10.1128/AEM.01228-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Detection of Salmonella enterica Subpopulations by Phenotype Microarray Antibiotic Resistance Patterns{triangledown} ,{dagger}

Jean Guard-Bouldin,1* Cesar A. Morales,1 Jonathan G. Frye,2 Richard K. Gast,1 and Michael Musgrove1

U.S. Department of Agriculture, ESQRU,1 U.S. Department of Agriculture, BEAR, 950 College Station Road, Athens, Georgia 306052

Received 1 June 2007/ Accepted 26 September 2007


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ABSTRACT
 
Three strains of Salmonella enterica serotype Enteritidis were compared to Salmonella enterica serotype Heidelberg, Salmonella enterica serotype Newport, and Salmonella enterica serovar Typhimurium for growth in the presence of 240 antibiotics arranged within a commercial high-throughput phenotype microarray. The results show that antibiotic resistances were different for subpopulations of serotype Enteritidis separated only by genetic drift.


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INTRODUCTION
 
Salmonella enterica serotype Enteritidis is the only one of over 1,400 Salmonella enterica serotypes that efficiently contaminates the contents of eggs and causes disease in humans (4, 15, 20). It is a model serotype for evaluating how genetic drift impacts phenotype, because mutational mapping of genetically related pathotypes has been completed. Current estimates of the genetic distances between serotype Enteritidis subpopulations, between serotype Enteritidis phage types, and between serotype Enteritidis and other Salmonella serotypes with a common lipopolysaccharide core structure are estimated at one single-nucleotide polymorphism for every 10,000, 1,000, and 100 contiguous base pairs, respectively. A rapid screening method that detects the subpopulation biology of strains that appear to be clonal would facilitate epidemiological investigations that monitor the emergence of genetic drift that impacts human health. To identify antibiotics for this purpose, we used a commercially available high-throughput phenotype microarray (Phenotype MicroArray [PM]; Biolog, Inc.) to compare the antibiotic resistance patterns of three subpopulations of serotype Enteritidis to those of three other serotypes of S. enterica, namely serotype Heidelberg, serovar Typhimurium, and serotype Newport. Specifically, the strains evaluated were (i) egg-contaminating, biofilm-positive serotype Enteritidis phage type 4 strain ESQRU 22079 (serotype Enteritidis PT4) (17); (ii) biofilm-positive serotype Enteritidis phage type 13a strain ESQRU 21027, which does not contaminate eggs (BF serotype Enteritidis PT13a) (18); (iii) wild-type egg-contaminating serotype Enteritidis PT13a strain ESQRU 21046, which does not produce biofilm (wt serotype Enteritidis PT13a) (18); (iv) serotype Heidelberg strain ESQRU 23018, a human isolate from a rare egg-associated outbreak (13); (v) serotype Newport strain ESQRU 24012, which has multiple antibiotic resistances; and (vi) serovar Typhimurium strain ESQRU 99187, which was isolated from the environment of chickens. Strains of these four serotypes were recovered from 50%, 53%, and 51% of laboratory-confirmed cases of salmonellosis in the United States for the years 2004, 2005, and 2006, respectively (26).


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PM analysis.
 
PM testing was performed by Biolog's PM Services group (Hayward, CA). The basic growth medium chemistry for PM analysis was published previously (3, 27). PM analysis was conducted in duplicate after incubation of the strains at 37°C for 48 h, and the data were analyzed by Student's t test to confirm that the results were uniform between runs. The metabolism of D-serine was an internal control, because BF serotype Enteritidis PT13a has a 10-bp deletion in the serine deaminase gene (dsdA), and the algorithm for defining when wells were otherwise positive for growth above baseline based on the respiratory activity (RA) of the cells in the initial inoculum has been described previously (18). Raw data were obtained for 10 96-well master plates (Biolog PM plates 11 to 20), which included 240 antibiotics arranged as a dilution series across four wells (960 wells in total). Data were recorded as RAs and were filtered in Microsoft Excel to identify statistically significant differences by Student's t test (see Table S1 in the supplemental material). Antibiotic susceptibilities were also tested by a conventional assay (Sensititre Microbiologic Systems, Westlake, OH) (12).


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Results.
 
The conventional assay indicated that serotype Newport had multiple antibiotic resistances and that wt serotype Enteritidis PT13a was resistant to ampicillin and tetracycline (Table 1). Filtering of all PM data indicated that the results for five ß-lactam antibiotics and sodium selenite showed significant (P < 0.001) differences between BF and wt serotype Enteritidis PT13a (Table 2). Wild-type serotype Enteritidis PT13a grew almost as well as serotype Newport in the ß-lactam antibiotics amoxicillin, carbenicillin, and penicillin G in the PM (Table 2): specifically, serotype Newport had average RAs of 329.6, 296.0, and 345.6, and wt serotype Enteritidis PT13a had average RAs of 270.4, 274.3, and 289.8 for these compounds, respectively (see Table S1 in the supplemental material) (18). Both wt serotype Enteritidis PT13a and serotype Newport grew in sodium selenite (RA > 50), whereas BF serotype Enteritidis PT13a did not (Table S1 in the supplemental material). Overall, sensitivity to the ß-lactam antibiotics differentiated between monomorphic strains of serotype Enteritidis as well as did the control compound D-serine, which links an easily observable phenotype to a known genomic lesion—a 10-bp deletion (bp 3880104 to 3880113 within the reference genome of serotype Enteritidis PT4). Two other findings for the compounds in Table 2 were that the monomorphic BF serotype Enteritidis PT13a strains were significantly different from all other strains and that wt serotype Enteritidis PT13a had resistances that were more similar to those of serotype Newport than could be detected by the conventional assay (Table 2). The sensitivity of wt serotype Enteritidis PT13a to amoxicillin-clavulanic acid in the conventional assay is more appropriately reported as intermediate, because values were eight times higher than those for any of the other Salmonella strains that were sensitive (Table 1).


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  		TABLE 1. Antibiotic resistance patterns of serotypes of S. enterica by sensititer assay


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  		TABLE 2. Differentiation of S. enterica subpopulations and serotypes by antibiotic compounds that differentiate between monomorphic subpopulations of serotype Enteritidis

Thirty-six compounds distinguished monomorphic PT13a strains from dimorphic serotype Enteritidis PT4 and from all other serotypes (Table 3). Serovar Typhimurium was somewhat unique, because its sensitivity was intermediate to that of monomorphic and dimorphic strains of serotype Enteritidis (Table 3). Serovar Typhimurium grew more like monomorphic serotype Enteritidis PT13a strains in 14 (38.9%) of the 36 compounds, whereas serotype Heidelberg and serotype Newport resembled monomorphic strains in their growth in only 8.3% and 11.1% of the compounds, respectively. These results suggest that serotype Heidelberg and serotype Newport grew more like dimorphic serotype Enteritidis PT4 than like the monomorphic strains.


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  		TABLE 3. Differentiation of S. enterica subpopulations and serotypes by antibiotic compounds that screen for dimorphism

The conventional assay for antibiotic resistances suggested that wt serotype Enteritidis PT13a was resistant to tetracycline. However, there was no evidence of this resistance in the PM (Table 4). In contrast, serotype Newport was resistant to tetracycline antibiotics (P < 0.01) in both the conventional and PM assays. The resistance of wt serotype Enteritidis PT13a to tetracycline thus appears to be overstated by the conventional assay. Differences in antibiotic concentrations and in the media used to assess resistance could account for the variation between assays.


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  		TABLE 4. Differentiation of S. enterica subpopulations and serotypes by the tetracycline class of antibiotic compounds


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Conclusions.
 
Several recent antibiogram studies of field isolates suggest that serotype Enteritidis is developing some antibiotic resistances (2, 5-11, 14, 16, 19, 21-25). However, genomic resequencing has not detected any differences in the mar regulon of strains of serotype Enteritidis. If the chromosome of serotype Enteritidis bacteria is currently evolving antibiotic resistances from small-scale evolutionary events, then an assay for subpopulation biology using antibiotics could help identify genetic drift that impacts public health (1). The results from these analyses improved the interpretation of resistances to the ß-lactam and tetracycline classes of antibiotics by combining information from both the PM and conventional assays. Overall, resistance to ß-lactam antibiotics was understated and resistance to the tetracycline class was overstated for serotype Enteritidis by the conventional assay.


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Microarray data sequence.
 
The sequence data for serotype Enteritidis PT4 were produced by the Pathogen Genome Sequencing Group at the Sanger Institute and can be obtained from ftp://ftp.sanger.ac.uk/pub/pathogens/Salmonella/SePT4.dbs. Complete mutational maps and partial confirmatory comparative genome resequencing data for BF and wt serotype Enteritidis PT13a are available at http://www.ncbi.nlm.nih.gov/genomes/static/Salmonella_SNPS.html.


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ACKNOWLEDGMENTS
 
This research was supported primarily by funds from ARS project number 6612-32000-004-00. Funds for the analysis of conventional resistance were provided by ARS project number 6612-32000-002-00.


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FOOTNOTES
 
* Corresponding author. Mailing address: U.S. Department of Agriculture, Agricultural Research Service, Egg Safety and Quality Research Unit, 950 College Station Road, Athens, GA 30605. Phone: (706) 546-3446. Fax: (706) 546-3035. E-mail: Jean.Guard.Bouldin{at}ars.usda.gov Back

{triangledown} Published ahead of print on 26 October 2007. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


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Applied and Environmental Microbiology, December 2007, p. 7753-7756, Vol. 73, No. 23
0099-2240/07/$08.00+0     doi:10.1128/AEM.01228-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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